Poxviruses encode enzymes and factors needed for replication of their genomes within the cytoplasm of infected cells. Vaccinia virus, the prototypic member of the poxvirus family, provides a unique system for combining biochemical and genetic approaches for investigating mechanisms of DNA replication. The DNA is synthesized as concatemers that are resolved into unit length genomes and packaged during virus assembly. Studies with conditional lethal mutants indicate that five VACV early proteins are required for DNA replication: namely E9 DNA polymerase, D4 uracil DNA glycosylase, A20 processivity factor, B1 protein kinase and D5 nucleoside triphosphatase (NTPase). The DNA polymerase catalyzes primer- and template-dependent synthesis and possesses 3 to 5 exonucleolytic activity. The essential role of D4 in DNA replication is independent of its uracil DNA glycosylase activity, which presumably has a facultative repair function. The A20 and D4 proteins interact and together provide processivity for the DNA polymerase. The B1 kinase was recently shown to phosphorylate a cellular DNA-binding protein called BAF and prevent the latter from blocking VACV DNA replication. The fast stop DNA replication phenotype of conditional lethal D5 mutants suggested a function at the replication fork. D5 also interacts with A20 and forms multimers. Potential roles for D5 have come from extensive protein sequence analyses, which indicate that the 90-kDa D5 protein is a member of the helicase superfamily III within the AAA+ class of NTPases, which includes the replicative helicases of some other DNA and RNA viruses.